Fig. 5.

Protein transduction of recombinant TAT-CRP2 into neonatal cardiomyocytes induces up-regulation of SMC genes. (A) Schematic representation of recombinant TAT fusion protein constructs. His-6, polyhistidine tag; PTD, protein transduction domain of HIV TAT protein; HA, epitope tag of influenza hemagglutinin protein. Recombinant TAT-PTD-CRP2 proteins (50 μg/ml) were added to tissue culture medium incubating freshly isolated neonatal cardiomyocytes. Protein transduction occurs in a rapid (<30 min), efficient (>97%), and concentration-dependent fashion (10–70 μg/ml). (B) Freshly isolated neonatal rat cardiomyocytes were plated overnight and then treated with recombinant TAT-CRP2 fusion protein. Whole-cell lysates were harvested at various time points, and Western blot analysis was performed to verify transduction of HA epitope-tagged TAT-CRP2. (C) Immnuofluorescent staining was carried out to detect coexpression of SMC marker genes (SMA, calponin, and SM-MHC) and cardiac-specific troponin T (TnT) in neonatal cardiomyocytes 3 d after TAT-CRP2 or TAT-β gal treatment. SMC contractile proteins were present in cardiomyocytes treated with TAT-CRP2 but not in the TAT-β gal control group. Indirect immunofluorescent staining was performed after 72 h of TAT fusion protein treatment. (D) The effect of recombinant TAT-CRP2 (Left) or TAT-β gal (Right) treatment on SM-MHC induction in cardiomyocyte assessed over time by semiquantitative RT-PCR (top three blots) and Western immunoblot analysis (bottom two blots). Arrow, time at which TAT-fusion proteins were added to the media. (E) The effect of cycloheximide (CHX) on TAT-CRP2-induced SM-MHC mRNA levels assessed by semiquantitative RT-PCR analysis. Cardiac myocytes were preincubated with cycloheximide (25 μg/ml) for 2 h before exposure to TAT proteins.